The long-range goal of this research is to understand the molecular mechanisms involved in the transcriptional regulation of a eukaryotic gene. To achieve this goal, we will analyze the control of transcription of yeast genes required for galactose utilization (the GAL genes), in which two key regulatory gene products (GAL4 and GAL80) control the inducibility of five GAL structural genes at the level of transcription. Three interrelated lines of research will be conducted. The first is focused towards identifying DNA sequences required for promoter function and proper regulation of three GAL structural genes. This involves analyzing the affects of mutations generated in vitro on GAL-lacZ fusions. The second line of research involves constructing strains which overproduce cloned GAL regulatory gene products (GAL4 and GAL80), followed by purification and characterization of these two proteins. The third line of research is directed towards developing a transcription system for transcribing yeast genes by RNA polymerase II in vitro. At all stages of the work, we plan to correlate the results of experiments performed in vivo with those performed in vitro. This will, hopefully, enable us to draw firm conclusions concerning molecular mechanisms of transcriptional control in eukaryotes.